1. Field of the Invention:
The invention is directed to a composition and a method for the determination of serum globulin.
2. Description of the Prior Art:
Globulins are simple proteins, present in blood, lymph and the cytoplasm of cells. They are usually associated with albumins, one or the other being predominant. They give all the ordinary protein tests and are coagulable by heat.
Globulins differ from the albumins, in that they are insoluble in pure (salt-free) water. However, they are soluble in neutral solutions of the salts of strong acids with strong bases, such as sodium chloride. Globulins require a certain concentration of salt in order to remain in solution. They precipitate when the concentration of salt is lowered by dilution of dialysis. In general the globulins are precipitated by half saturation of their solutions with ammonium sulfate, i.e., by the addition to their solutions of an equal volume of saturated ammonium sulfate solutions. Most globulins are also precipitated from their solutions by saturation with solid sodium chloride or magnesium sulfate.
Blood serum contains a variety of globulins characterized by differences in solubility, in precipitability by ammonium or sodium sulfate, and the in rate of electrophoretic migration.
Colorimetry and photometry have been widely applied in the field of clinical chemistry for the determination of albumin and globulin components in serum and plasma and the diagnosis of disease states. Not all proteins contain the same amino acids, and for this reason color tests give reactions varying in intensity according to the nature and the amount of the groups contained in the particular protein under examination.
One of the oldest tests known for the determination of serum proteins is the so-called biuret test. This test is given by those substances whose molecules contain two carbamyl groups joined either directly together or through a single atom of nitrogen or carbon. The test derives its name from the fact that biuret, which is formed on heating urea to 180.degree. C., responds to the test. Proteins respond positively because their molecules include pairs of aminocarboxyl groups.
Since the biuret reaction determines total serum protein, the albumin and globulin fractions may be determined indirectly by salt fraction or by electrophoresis. In salt fractionation, globulin is precipitated by adding salt solution leaving albumin in solution. The protein concentrations of the unfractionated sample and of the albumin solution are determined by the biuret reaction and the globulin concentration is calculated from the difference of the two. In the electrophoretic method, serum protein is separated into different fractions using starch, gel, agrigel, paper or cellulose acetate electrophoresis. The fractions are strained and their relative proportions are determined. The concentration of different fractions may be determined from these relative proportions and the total protein concentration of the sample determined by biuret reaction. Neither of the above-mentioned methods is entirely satisfactory for routine clinical purposes since both require multiple manipulations.
Albumin and globulin may also be assayed by colorimetric determination of albumin through selective dye-binding with 2-(4'-hydroxyazobenzene)benzoic acid (HABA), bromcresol green or methyl orange and calculating globulin by the difference from total protein. The selective dye-binding method is subject to numerous interference in specie variations, and determinations made by this method are subject to many innacuracies.
Globulin may be directly determined by the well known Hopkins-Cole reaction which is a colorimetric method dependent upon the presence of the tryptophane group in the protein. The tryptophane residue reacts with glyoxalic acid and concentrated sulfuric acid to give a purple color.
Goldenberg (U.S. Pat. No. 3,607,081) developed a reagent for the determination of globulins in biological fluid comprising a solution of glyoxalic acid and cupric sulfate pentahydrate in a mixture of glacial acetic and sulfuric acids. One of the disadvantages of the Goldenberg composition is its requirement for extremely high quantities of the acetic acid component, i.e., from about 80 to about 99 parts per volume.
Neeley et al (Clin. Biochem. 8, pp. 273-278, 1975) developed another reagent for the same purpose which contains glyoxalic acid, copper sulfate, concentrated sulfuric acid and lactic acid. As in the case of Goldenberg composition, the Neeley et al reagent is based on the Hopkins-Cole reaction.
A singular disadvantage of the Goldenberg and the Neeley et al compositions is that both reagents develop considerable interfering color in the presence of albumin. Note Example VI, below.
In U.S. Pat. No. 3,558,516, Wybenga discloses a reagent for the determination of cholesterol in biological fluids which comprises ferric perchlorate in ethyl acetate and sulfuric acid. Similarly, in U.S. Pat. No. 3,001,950 Hopper discloses a mixture of sulfuric acid, acetic anhydride and acetic acid for the same purpose.
The present invention differs from the Wybenga composition in that it contains a critically minimal volume of water. The presence of water is essential for the reaction with globulin to take place, and when present in sufficient quantity, it will completely prevent reaction of cholesterol with the reagent. This is especially true when the added volume of water is at least 15% v/v concentration. Example VIII below shows the non-reactivity of the novel composition with cholesterol and the identity of results obtained against sera with and without added cholesterol.
In general, none of the prior art procedures has been entirely satisfactory. It would be desirable to develop a stable reagent for globulin determination which would contain lower proportions of acid and would produce little or no interfering color when reacted in the presence of albumin. It would also be desirable to develop a method which would give results comparable to that of electrophoretic analyses or salt precipitation in serum of different species.